An aspartyl protease defines a novel pathway for export of Toxoplasma proteins into the host cell

Toxoplasma gondii is an obligate intracellular Apicomplexan parasite capable of invading and surviving within nucleated cells in most warm-blooded animals. This remarkable task is achieved through the delivery of effector proteins from the parasite into the parasitophorous vacuole and host cell cytosol that rewire host cellular pathways, facilitating parasite evasion of the immune system. Here, we have identified a novel export pathway in Toxoplasma that involves cleavage of effector proteins by the Golgi-resident aspartyl protease 5 (ASP5) prior to translocation into the host cell. We demonstrate that ASP5 cleaves a highly constrained amino acid motif that has some similarity to the PEXEL motif of Plasmodium parasites. We show that ASP5 can mature effectors at both the N- and C-terminal ends of proteins and is also required for the trafficking of proteins without this motif. Furthermore, we show that ASP5 controls establishment of the nanotubular network and is required for the efficient recruitment of host mitochondria to the parasitophorous vacuole membrane. Global assessment of host gene expression following infection reveals that ASP5-dependent pathways influence thousands of the transcriptional changes that Toxoplasma imparts on its host cell. This work characterizes the first identified machinery required for export of Toxoplasma effectors into the infected host cell. Overall design: Three groups of human foreskin fibroblasts are compared. Each group has 3 replicates giving a total of 9 samples. The first group of samples are infected with wild type (GRA16HA) Toxoplasma gondii, the second group with Asp5 knock-out Toxoplasma gondii, and the final group remain uninfected. All fibroblasts are generated from one donor sample.

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Coffey MJ, Sleebs BE, Uboldi AD, Garnham A, Franco M, Marino ND, Panas MW, Ferguson DJ, Enciso M, O'Neill MT, Lopaticki S, Stewart RJ, Dewson G, Smyth GK, Smith BJ, Masters SL, Boothroyd JC, Boddey JA, Tonkin CJ